Preserving and re-shaping the genome: the role of homologous recombination in DNA repair and replication
Co-workers: Nadiya Ishnazarova, Manisha Jalan, Carl Morrow, Fikret Osman, Sanjeeta Tamang
My group studies how homologous recombination repairs DNA double-strand breaks and processes perturbed replication forks. By understanding these fundamental processes we aim to discover how their dysfunction causes diseases such as cancer.
Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) and damaged replication forks, and as such is critical for maintaining genome integrity. However, its injudicious use can result in potentially harmful genome rearrangements. Cells therefore have to carefully control when and how HR is used. The importance of this to human health is underscored by a number of diseases such as familial breast cancer, Fanconi anaemia, and Bloom’s and Werner’s syndromes that are linked to dysfunctional HR. These diseases variously manifest clinical features such as cancer proneness, immunodeficiency, developmental problems and premature ageing. HR is also essential in most eukaryotes for promoting the correct segregation of homologous chromosomes during the first meiotic division. A failure in HR in the human oocyte can result in aneuploidy, which is a factor in many spontaneous pregnancy losses, and in the case of trisomy 21 results in Down’s syndrome.
Homologous recombination and DSB repair
Pivotal to most HR is the assembly of a long polymer of Rad51 onto a tail of single-stranded DNA (generated by the 5' - 3' resection of a DSB) to form a nucleoprotein filament that first locates an intact homologous donor sequence, and then invades it. Strand invasion generates a displacement loop (D-loop) at which DNA synthesis can be primed from the invading 3' end. Recombination can then proceed by one of several different pathways (see Figure 1). These variously give rise to two types of recombinant product - crossovers (COs), where the DNA flanking the site of recombination is reciprocally exchanged between the recombining molecules, and non-crossovers (NCOs), where the flanking DNAs maintain their starting configuration. COs are essential during meiosis for the establishment of chiasmata that help guide correct chromosome segregation. However, in mitotic cells crossing over between homologous chromosomes can result in loss of heterozygosity (LOH), and between ectopic homologous sequences, gross chromosomal rearrangements (GCRs; e.g. translocations, deletions, duplications and inversions). Both LOH and GCRs are common features of diseases such as cancer. It is therefore clear that HR has to be carefully controlled so that it is used in the appropriate way, at the appropriate time and appropriate place.
A key part of HR promotion and control acts through the processing of recombination intermediates. For example, proteins that disrupt the Rad51 nucleofilament can abort HR, whereas those that function on DNA intermediates, which derive from strand invasion (i.e. D-loops and Holliday junctions (HJs)), determine whether a CO or NCO is generated (Figure 1). We have identified a number of such proteins (including the DNA helicases Fbh1, Fml1, Rqh1 and Srs2, and the endonuclease Mus81-Eme1) in the model eukaryote Schizosaccharomyces pombe (fission yeast), whose human orthologues have disease associations. A major goal at the moment is to understand exactly how these proteins are governed to give the right balance of COs and NCOs in mitotic and meiotic cells.
Homologous recombination and DNA replication
Before a cell divides it has to replicate its genetic material so that each new daughter cell can receive a copy. This process of DNA replication is performed by complex protein machines, which are assembled onto DNA at chromosomal sites known as replication origins. During S-phase multiple origins on every chromosome fire, each releasing a pair of replication forks that travel in opposite directions away from their origin. DNA replication is completed when forks from adjacent origins merge. The failure of even a single pair of forks to merge results in a region of unreplicated DNA that can lead to DNA breakage and ultimately genetic mutations that cause diseases such as cancer. The many proteins that bind chromosomal DNA threaten successful completion of DNA replication by physically impeding the progress of the replication fork. Elaborate mechanisms enable replication forks to overcome such roadblocks and safely and accurately merge during replication termination. Our goal is to elucidate these mechanisms by discovering how failed replication forks are disassembled at barriers, understanding how HR restarts blocked forks, and determining what prevents pathological replication termination.
- Nguyen M.O., Jalan M., Morrow C.A., Osman F. and Whitby M.C. (2015) Recombination occurs within minutes of replication fork blockage by RTS1 producing restarted forks that are prone to collapse. eLife, e04539
- Lorenz A., Mehats A., Osman F. and Whitby M.C. (2014) Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination. Nucleic Acids Research, 42, 13723-13735
- Steinacher R., Osman F., Dalgaard J.Z., Lorenz A. and Whitby M.C. (2012) The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability. Genes and Development, 26, 594-602
- Lorenz A., Osman F., Sun W., Nandi S., Steinacher R. and Whitby M.C. (2012) The fission yeast FANCM ortholog directs non-crossover recombination during meiosis. Science, 336, 1585-1588
- Yan Z., Delannoy M., Ling C., Daee D., Osman F., Muniandy P.A., Shen X., Oostra A.B., Du H., Steltenpool J., Lin T., Schuster B., Decaillet C., Stasiak A., Stasiak A.Z., Stone S.,Hoatlin M.E., Schindler D., Woodcock C., Joenje H., Sen R., de Winter J.P., Li L., Seidman M.M., Whitby M.C., Myung K., Constantinou A. and Wang W. (2010) A novel histone-fold complex and FANCM form a conserved DNA remodeling complex to maintain genome stability. Molecular Cell, 37, 865-878
- Lorenz A., Osman F., Folkyte V., Sofueva S. and Whitby M.C. (2009) Fbh1 limits Rad51-dependent recombination at blocked replication forks. Molecular and Cellular Biology 29, 4742-4756
- Sun W., Nandi S., Osman F., Ahn J., Jakovleska J., Lorenz L. and Whitby M.C. (2008) The fission yeast FANCM ortholog Fml1 promotes recombination at stalled replication forks and limits crossing over during double-strand break repair. Molecular Cell, 32, 118-128
Figure 1: DSB repair pathways in fission yeast
Figure 2: How a replication fork barrier can cause genome instability
Figure 3: Visualizing the recruitment of a recombination protein (Rad52) to a replication fork blocked at a site-specific barrier in a live cell. Rad52 is tagged with yellow fluorescent protein (YFP), and the replication fork barrier is marked by the LacI repressor fused to tdKatushka2. PCNA fused to cyan fluorescent protein (CFP) is also imaged.
Figure 4: Replication fork blockage at a site-specific barrier detected by native two-dimensional gel electrophoresis
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